High sensitivity temporal focusing widefield multiphoton endoscope capable of deep imaging

Abstract

An imaging system is provided that includes a pulsed light source providing pulsed light and is applicable to both microscopes and endoscopes. One or more optical elements with certain dispersive properties are positioned to receive the pulsed light and apply selective dispersive properties to shift the focal plane according to the user and to produce two photon (2p) wide field uniform illumination and 2p wide field structured illumination for the purpose of improving the optical axial resolution and rejection of background signal. An imaging element receives the signal arising from the 2p wide field uniform illumination and 2p wide field structured illumination and produces a respective 3D resolved image of a sample.

What is claimed is: 
     
       1. An imaging system comprising:
 a pulsed light source that provides pulsed light; 
 one or more dispersive elements that are positioned to receive the pulsed light and apply selective dispersive properties to recombine the spectrally dispersed pulse at a certain position along the optical axis where with the recombined pulse, a sequence of two photon (2p) wide field uniform illumination and 2p wide field structured illumination is generated at the focal plane; and 
 an imaging element that receives multiple 2p wide field uniform illumination and 2p wide field structured illumination images to produce respective 3D resolved images of a sample, wherein the group velocity dispersion (GVD) of the pulsed light is tuned to induce a quadratic spectral phase, which effectively shifts the focal plane and thereby allows scanning in the axial direction of the pulsed light. 
 
     
     
       2. The imaging system of  claim 1 , wherein the pulsed light source comprises a laser and a beam expander of variable magnification. 
     
     
       3. The imaging system of  claim 1 , wherein the one or more dispersive elements disperses the pulsed light to form a spectrum. 
     
     
       4. The imaging system of  claim 1 , wherein the image element comprises an objective which collimates each spectral component associated with the uniform illumination and 2p wide field illumination and recombines them at a focal plane where the original pulse width of the pulsed light is restored. 
     
     
       5. The imaging system of  claim 1 , wherein the pulsed light are positioned at a focal plane thereby generating high peak power and increasing the probability of a multiphoton process occurring while maintaining a wide field of view. 
     
     
       6. The imaging system of  claim 1 , wherein the 2p wide field illumination permits axial scanning at the operator end of an endoscope, which reduces the size of the endoscope head. 
     
     
       7. The imaging system of  claim 1 , wherein the 2p wide field illumination permits axial scanning at the operator end of an endoscope through the control of the spectral phase of the input pulsed beam. 
     
     
       8. The imaging system of  claim 1 , wherein the image element comprises a detector being located within an endoscope head allowing the signal-to-noise ratio to increase. 
     
     
       9. A method for performing deep tissue imaging through improving the optical sectioning as well as the rejection of background signal comprising:
 providing a pulsed light source that provides pulsed light; 
 positioning one or more dispersive elements to receive the pulsed light and apply selective dispersive properties to recombine the spectrally dispersed pulse at a certain position along the optical axis where with the recombined pulse a sequence of two photon (2p) wide field uniform illumination and 2p wide field structured illumination is generated at the focal plane; and 
 receiving multiple 2p wide field uniform illumination and 2p wide field structured illumination images to produce respective 3D resolved images of a sample using an imaging element, wherein the group velocity dispersion (GVD) of the pulsed light is tuned to induce a quadratic spectral phase, which effectively shifts the focal plane and thereby allows scanning in the axial direction of the pulsed light. 
 
     
     
       10. The method of  claim 9 , wherein the pulsed light source comprises a laser and a beam expander. 
     
     
       11. The method of  claim 9 , wherein the one or more dispersive elements disperses the pulsed light to form a spectrum. 
     
     
       12. The method of  claim 9 , wherein the image comprises using images formed using the 2p wide field uniform illumination and 2p wide field structured illumination. 
     
     
       13. The method of  claim 9 , wherein the imaging element comprises focusing the dispersed spectrum at the back focal plane of an objective, which collimates each spectral component associated with the 2p wide field uniform illumination and 2p wide field structured illumination and recombines them at a focal plane where the original pulse width of the pulsed light is restored. 
     
     
       14. The method of  claim 9 , wherein the pulsed light are positioned at a focal plane thereby generating high peak power and increasing the probability of a multiphoton process occurring while maintaining a wide field of view. 
     
     
       15. The method of  claim 9 , wherein the 2p wide field illumination permits axial scanning at the operator end of the method, which reduces the size of the method head. 
     
     
       16. The method of  claim 9 , wherein the image element comprises a detector being located within the method head allowing the signal-to-noise ratio to increase. 
     
     
       17. The method of  claim 9 , wherein the 2p wide field uniform illumination and 2p wide field structured illumination are capable of being used as background rejection techniques to improve optical sections in the axial direction. 
     
     
       18. The method of  claim 9  further comprising combining the multiple uniform wide-field 3D resolved two-photon illumination images and multiple structured wide-field 3D resolved two-photon illumination images for rejection of out-of-focal noise from in-focus signal using a computer algorithm. 
     
     
       19. The method of  claim 9 , wherein a computational algorithm of photon reassignment based on maximum likelihood estimation applied for 3D image reconstruction from a z-stack of uniform and structured wide-field 3D resolved two-photon illumination images to improve image stack signal-to-noise (SNR) level. 
     
     
       20. The method of  claim 9 , wherein the structured illumination is periodically and spatially shifted in time. 
     
     
       21. The method of  claim 20 , wherein the structured illumination is lock-in detected at the period of the shifting of the structured light pattern allowing shot-noise limited detection of structured illumination imaging increasing its contrast.